A novel probe to assess cytosolic entry of exogenous proteins.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA.

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Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA. peter.cresswell@yale.edu.

Abstract

Dendritic cells use a specialized pathway called cross-presentation to activate CD8+ T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem. Here we describe the development of a sensitive reporter substrate, an N-glycosylated variant of Renilla luciferase fused to the Fc region of human IgG1. The luciferase variant is designed to be enzymatically inactive when glycosylated, but active after the asparagine to aspartic acid conversion that occurs upon deglycosylation by the cytosolic enzyme N-glycanase-1. The generation of cytosolic luminescence depends on internalization, deglycosylation, the cytosolic AAA-ATPase VCP/p97, and the cytosolic chaperone HSP90. By incorporating a T cell epitope into the fusion protein, we demonstrate that antigen dislocation into the cytosol is the rate limiting step in cross-presentation.

Dislocation kinetics of ddRLuc-Fc in 239T-FcRγIIA-Kb cells. 293T-FcRγIIA-Kb cells were incubated with ddRLuc-Fc-bound 3 μm latex beads for phagocytosis (a) or 100 μg mL-1 soluble ddRLuc-Fc for endocytosis (b) at 37 °C in the presence or absence of various drugs (200 nM Epox, 20 μm zVAD, or 32 μm Rad). Cell lysates were prepared and analyzed as described in Fig. at each time point, and RLU is represented as the percent of maximum value. Points represent the mean +/−s.d. of one representative experiment with duplicates. Representative data of three independent experiments are shown

Correlation between antigen dislocation and T cell activation. a Schematic description of ddRLuc-FcOVA. b, c The 293T-FcRγIIA-Kb cells were fed ddRLuc-FcOVA-bound 3 μm latex beads for phagocytosis and incubated at 37 °C for 8 h. For measuring luciferase activity, total cell lysate was prepared, processed, and analyzed as described in Fig. . The percent RLU values shown are generated from samples treated with DMSO or a combination of Epox plus various inhibitors relative to Epox-treated samples (set to 100%). Cross-presentation was assayed using IL-2 secretion by the B3Z hybridoma as described in Methods. The percent cross-presentation shown corresponds to the amount of IL-2 released from the B3Z hybridoma activated by the cells treated with various inhibitors relative to DMSO-treated samples (set to 100%). b Opposing effects of proteasome inhibition by 200 nM Epox on dislocation and cross-presentation. Bars represent the mean +/−s.d. of five independent experiments per treatment, and numbers indicate the mean value. c The correlation between antigen dislocation and cross-presentation was plotted by incorporating data from treatments with different concentrations of chloroquine (CHQ), CB5083, and cytochalasin D (Cyto D) plus Dynasore (Dyn) in the presence (for dislocation) or absence (for cross-presentation) of Epox. Data from Supplementary Fig. were also incorporated into this figure. Points represent the mean +/−s.d. (for both x and y values) of one representative experiment with triplicates. Representative data from three independent experiments are shown